The field of gene therapy has made significant gains in recent years. The combination of genetic defects being identified and gene target/delivery methods being developed has led to an explosion in the number of clinical gene therapy protocols. The central focus of gene therapy is to develop methods for introducing new genetic material into somatic cells. To date two general classes of gene transfer methods have evolved. The first is DNA-mediated gene transfer and involves direct administration of DNA to the patient in various formulations. These methods use genes as medicines in a manner much like conventional organic or protein compounds. DNA-mediated gene transfer however has proven quite difficult. Methodology such as micro-injection, lipofection, and receptor mediated endocytosis have usually resulted in lower gene transfer, and have usually established only transient residence of the novel gene in the targeted cell. Permanent incorporation of genes into cells occurs rarely after DNA-mediated gene transfer in cultured cells (less than 1×105 cells) and has not been significantly observed in vivo. Thus DNA-mediated gene transfer may be inherently limited to the use of genes as medicines that are administered by conventional parenteral routes to provide a therapeutic effect over predictable period of time. Studies of a therapeutic gene product may be constituted by repetitively dosing the patient with degenerate material much like conventional pharmaceutical medicines.
Viral gene transfer on the other hand involves construction of synthetic virus particles (vectors) that lack pathogenic functions. The virus particles are incapable of replication and contain a therapeutic or diagnostic gene within the viral genome which is delivered to cells by the process of infection. To date the viral vector which has achieved the most success is the retroviral vector. The prototype for a retroviral mediated gene transfer is a retroviral vector derived from Moloney Murine Leukemia Virus. Retroviral vectors have several properties that make them useful for gene therapy. First is the ability to construct a “defective” virus particle that contains the therapeutic gene and is capable of infecting cells but lacks viral genes and expresses no viral gene products which helps to minimize host response to potential viral epitopes.
Retroviral vectors are capable of permanently integrating the genes they carry into the chromosomes of the target cell. Considerable experience in animal models and initial experience in clinical trials suggest that these vectors have a high margin of safety.
Vectors based on adenovirus have recently proven effective as vehicles for gene transfer in vitro and in vivo in several cell types. Adenoviral vectors are constructed using a deleted adenoviral genome that lacks either the e-3, e-4 or gene region and/or the e-1 gene region that is required for producing a replicating adenovirus particle. Recombinant genes are inserted into the site of the deleted gene region(s). Adenoviral particles are then produced in a cell line that is able to express e-1, e-4 or e-3 genes and thus capable of assembling a viral particle which contains only the recombinant viral genome with the therapeutic gene.
Adenoviral vectors differ from retroviral vectors in that they do not integrate their genes into the target cell chromosome. Adenoviral vectors will infect a wide variety of both dividing and non-dividing cells in vitro and in vivo with a high level of efficiency providing expression of their recombinant gene for a period of several weeks to months.
Current technology has enabled construction of adenoviral vectors that are incapable of proliferating however they are not completely “defective” and will express a series of viral gene products which can generate host immune response to the viral epitopes presented causing quick elimination of the already transient vector.
Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M. S., “Adenoviridae and Their Replication,” in Virology, 2nd ed., Fields et al., eds., Raven Press, New York 1990). The viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication.
Recombinant adenoviruses have several advantages for use as gene transfer vectors, including tropism for both dividing and non-diving cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Ther. 1:51-64, 1994).
The cloning capacity of an adenoviral vector is proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, and the deletion of a genomic region such as E1 whose function may be restored in trans from 293 cells (Graham, F. L., J. Gen. Virol. 36:59-72, 1977) or A549 cells (Imler et al., Gene Ther. 3:75-84, 1996). Such E1-deleted vectors are rendered replication-defective. The upper limit of vector DNA capacity is about 105%-108% of the length of the wild-type genome. Further adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g., complementation of E2 (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak et al, Hum. Gene Ther. 6:1575-1586, 1995). Maximum carrying capacity can be achieved using adenoviral vectors deleted for all viral coding sequences (Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
Transgenes that have been expressed to date by adenoviral vectors include p53 (Wills et al., Hum. Gene Ther. 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et al., Hum. Gene Ther. 5:979-985, 1994; omithine transcarbamylase (Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256, 1990; We et al., J. Biol. Chem. 271:3639-3646, 1996); adenosine deaminase (Mitani et al., Hum. Gene Ther. 5:941-948, 1994); interleukin-2 (Haddada et al., Hum. Gene Ther. 4:703-711, 1993); and α1-antitrypsin (Jaffe et al., Nature Genetics 1:372-378, 1992); thrombopoictin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).
The tropism of adenoviruses for cells of the respiratory tract has particular relevance to the use of adenovirus in Gene Ther. for cystic fibrosis (CF), which is the most common autosomal recessive disease in Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disturb the cAMP-regulated C1˜ channel in airway epithelia result in pulmonary dysfunction (Zabner et al., Nature Genetics 6:75-83, 1994). Adenoviral vectors engineered to carry the CFTR gene have been developed (Rich et al., Hum. Gene Ther. 4:461-476, 1993) and studies have shown the ability of these vectors to deliver CFTR to nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the airway epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:L75-83, 1994), and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies have shown that administering an adenoviral vector containing a DNA sequence encoding CFTR to airway epithelial cells of CF patients can restore a functioning chloride ion channel in the treated epithelial cells (Zabner et al., J. Clin. Invest. 97:1504-1511, 1996).
Modifications to the adenovirus genomic sequences contained in the recombinant vector have been attempted in order to decrease the host immune response (Yang et al., Nature Genetics 7:362-369, 1994; Lieber et al., J. Virol. 70:8944-8960, 1996; Gorziglia et al., J. Virol. 70:4173-4178; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
In addition to deletions in the adenovirus El region, first-generation adenoviral vectors often contain modifications to the E3 region in order to increase the packaging capacity of the vectors and to reduce viral gene expression (Yang et al., J. Virol. 69:2004-2015, 1995; Zsengeller et al., Hum. Gene Ther. 6:457-467, 1995; Brody et al., Hum. Gene Ther. 5:821-836, 1994). However, the adenovirus E3 regions contains certain proteins which modulate the host's antiviral immune response. The E3 transcription unit encodes the 12.5K, 6.7K, gp19K, 11.6K, 10.4K, 14.5K and 14.7K proteins (Wold et al., Trends Microbiol. 2:437-443, 1994). The E3 14.7K, 14.5K, and 10.4K proteins are able to protect infected cells from TNF-induced cytolysis. The adenovirus E3 gpl9K protein can complex with MHC Class 1 antigens and retain them in the endoplasmic reticulum, which prevents cell surface presentation and killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al., Trends Microbiol. 437-443, 1994), suggesting that its presence in a recombinant adenoviral vector may be beneficial. The E3 11.6K gene (Adenovirus death protein) is required for cell lysis and the release of adenovirus from infected cells (Tollefson et al., J. Virol. 70:2296-2306, 1996; Tollefson et al., Virology 220:152-162, 1996).
Earlier designs of adenoviral vectors in which the E3 region was modified have shown only transient expression of a transgene in the lungs of test animals (Yang et al., J. Virol. 69:2004-2015; Zsengeller et al., Hum Gene Ther. 6:457-467, 1995).
Modifications to the adenovirus E4 region have been introduced into adenoviral vectors in order to reduce viral gene expression and to further increase carrying capacity (Armentano et al., Hum. Gene Ther. 6:1343-1353, 1995). However, experiments in which adenoviral vectors were introduced into nude mice demonstrated that the context of the adenovirus E4 genomic region was a determinant in the persistence of expression, especially when the CMV promoter was used to control expression of the transgene (Kaplan et al., Hum. Gene Ther. 8:45-56, 1997; Armentano et al., J. Virol. 71:2408-2416, 1997).
The current state of adenoviral vector as well as viral vector based gene delivery requires the development of novel adenoviral vectors which demonstrate a capability for persistence and sustained expression of a transgene.